CN115348821A

CN115348821A - Novel nutrient for enhancing load-induced muscle hypertrophy

Info

Publication number: CN115348821A
Application number: CN202180020509.0A
Authority: CN
Inventors: 基思·巴尔; 安德鲁·菲利普; 西蒙·申克
Original assignee: University of Birmingham; University of California
Current assignee: University of Birmingham; University of California
Priority date: 2020-03-10
Filing date: 2021-01-15
Publication date: 2022-11-15
Also published as: WO2021072450A3; CA3175049A1; BR112022017939A2; US11312695B2; WO2021072450A2; EP4117455A2; US20210214329A1; JP2023517997A

Abstract

Methods and compositions are provided for increasing muscle hypertrophy, for example, by administering a novel combination of natural products that inhibit SIRT1.

Description

Novel nutrient for enhancing load-induced muscle hypertrophy

Cross Reference to Related Applications

This application claims priority from U.S. provisional patent application No. 62/987,807, filed 3/10/2020, which is incorporated herein by reference in its entirety.

Background

Muscle mass and strength are important aspects of human health, as mortality in individuals is associated with low muscle mass (17) and strength (14, 15). Low muscle mass and function also limits post-operative recovery and activity and increases the impact or risk of diseases such as diabetes, cardiovascular disease and cancer (2). Therefore, improving muscle mass and strength is a key component of long life (8).

Muscle mass and strength are also important components of human aesthetics and ability. Every year, a great deal of money is spent on supplements that purportedly result in increased muscle mass and strength. Many of these products are of great scientific interest because large-scale screening of products leading to improved muscle mass and strength is rare. One clinically effective method of increasing muscle mass gain as a result of training is to combine strength training with protein supplementation (3). However, very few other scientifically proven nutritional approaches have been reported to increase muscle mass in response to exercise.

Sirtuin 1 (SIRT 1) is an NAD + dependent deacetylase, which is activated in muscle in response to changes in cellular energy flux. It is known that metabolic stress during calorie restriction (6) and endurance exercise (3) directly activates SIRT1. Since calorie restriction and endurance exercise are also known to slow muscle growth (1, 7), we hypothesize that SIRT1 inhibits muscle growth. Protein acetylation is also associated with muscle growth. Ribosomal S6 protein kinase (S6K 1), the phosphorylation and activity of which has previously been shown to be associated with increased muscle protein synthesis and muscle hypertrophy (1), can be acetylated by the acetyltransferase p300 and deacetylated by SIRT1 (9). Almost every protein in the ribosome is regulated by acetylation, in addition to S6K1 (4). These data suggest that acetylation may be a novel method of modulating protein synthesis. Since load and nutrition lead to a transient increase in muscle protein synthesis thought to play an important role in muscle growth, we sought to determine whether altering acetylation could increase the increase in muscle fiber cross-sectional area in response to a hypertrophic stimulus.

Thus, there is a need in the art for new, scientifically sound, effective and safe methods and compositions for promoting muscle hypertrophy and increasing muscle mass and strength. The present application satisfies this need and provides other advantages as well.

Summary of The Invention

In one aspect, the present application provides a method of enhancing skeletal muscle growth in a mammal experiencing a muscle load comprising administering to the mammal a composition comprising a therapeutically effective amount of: (i) One or more catechins and (ii) tripterine or a derivative thereof.

In some embodiments, the one or more catechins include epicatechin mono-gallate or epigallocatechin-3-mono-gallate. In some embodiments, the one or more catechins include epicatechin mono-gallate and epigallocatechin-3-mono-gallate. In some embodiments, the tripterine derivative is dihydrotripterine. In some embodiments, the composition comprises epicatechin mono-gallate, epigallocatechin-3-mono-gallate, and tripterine. In some embodiments, the composition results in an increase in muscle fiber cross-sectional area of at least one skeletal muscle in the mammal. In some embodiments, the composition does not substantially alter the body weight or heart or liver weight of the mammal. In some embodiments, the composition reduces SIRT1 activity in one or more muscles of a mammal. In some embodiments, the composition increases acetylation of one or more ribosomal proteins in one or more muscles of the mammal.

In some embodiments, the composition is administered orally to the mammal. In some embodiments, the composition is formulated as a nutritional supplement or food additive. In some embodiments, the nutritional supplement or food additive is a pill, tablet, capsule, liquid, powder, energy bar, protein bar, or gel. In some embodiments, the mammal is a human. In some embodiments, the composition is formulated and administered such that the mammal receives from about 0.7 to 1.3 mg/kg/day of epicatechin mono gallate. In some embodiments, the composition is formulated and administered such that the mammal receives about 0.7 mg/kg/day of epicatechin mono gallate. In some embodiments, the composition is formulated and administered such that the mammal receives about 6-20 mg/kg/day of epigallocatechin-3-mono-gallate. In some embodiments, the composition is formulated and administered such that the mammal receives about 20 mg/kg/day of epigallocatechin-3-mono gallate. In some embodiments, the composition is formulated and administered such that the mammal receives about 0.2-0.5 mg/kg/day of tripterine. In some embodiments, the composition is formulated and administered such that the mammal receives about 0.5 mg/kg/day of tripterine. In some embodiments, the composition is formulated and administered such that the mammal receives about 0.7 mg/kg/day of epicatechin mono-gallate, about 20 mg/kg/day of epigallocatechin-3-mono-gallate, and about 0.5 mg/kg/day of tripterine. In some embodiments, the composition is formulated and administered such that the relative weight ratio of each of epicatechin mono-gallate, epigallocatechin-3-mono-gallate and celastrol received by the mammal is about 0.7. In some embodiments, the composition is formulated and administered such that the relative molar ratio of each of epicatechin mono-gallate, epigallocatechin-3-mono-gallate and tripterine received by the mammal is about 1.6. In some embodiments, the method further comprises increasing caloric intake and/or muscle growth promoting amino acid intake in the mammal concurrently with administration of the composition and muscle load. In some embodiments, the composition further comprises leucine, branched chain amino acids, or proteins with high leucine content.

In another aspect, the present disclosure provides a composition for enhancing muscle growth in a mammal undergoing muscle loading, the composition comprising a therapeutically effective amount of (i) one or more catechins and (ii) tripterine or a derivative thereof.

In some embodiments, the one or more catechins include epicatechin mono-gallate or epigallocatechin-3-mono-gallate. In some embodiments, the one or more catechins include epicatechin mono-gallate and epigallocatechin-3-mono-gallate. In some embodiments, the tripterine derivative is dihydrotripterine. In some embodiments, the composition comprises epicatechin mono-gallate, epigallocatechin-3-mono-gallate, and tripterine. In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is formulated as a nutritional supplement or food additive. In some embodiments, the nutritional supplement or food additive is a pill, tablet, capsule, liquid, powder, energy bar, protein bar, or gel. In some embodiments, the composition further comprises leucine, branched chain amino acids, or proteins with high leucine content.

In some embodiments, the composition is formulated such that the mammal receives about 0.7-1.3 mg/kg/day of epicatechin mono gallate. In some embodiments, the composition is formulated such that the mammal receives about 0.7 mg/kg/day of epicatechin mono gallate. In some embodiments, the composition is formulated such that the mammal receives about 6-20 mg/kg/day of epigallocatechin-3-mono-gallate. In some embodiments, the composition is formulated such that the mammal receives about 20 mg/kg/day of epigallocatechin-3-mono gallate. In some embodiments, the composition is formulated such that the mammal receives about 0.2-0.5 mg/kg/day of tripterine. In some embodiments, the composition is formulated such that the mammal receives about 0.5 mg/kg/day of tripterine. In some embodiments, the composition is formulated such that the mammal receives about 0.7 mg/kg/day of epicatechin mono-gallate, about 20 mg/kg/day of epigallocatechin-3-mono-gallate, and about 0.5 mg/kg/day of tripterine. In some embodiments, the relative weight ratio of each of epicatechin mono-gallate, epigallocatechin-3-mono-gallate and celastrol in the composition is about 0.7. In some embodiments, the relative molar ratio of epicatechin mono-gallate, epigallocatechin-3-mono-gallate and celastrol, respectively, in the composition is about 1.6.

Other objects, features and advantages of the present application will be apparent to those skilled in the art from the following detailed description and the accompanying drawings.

Brief description of the drawings

Fig. 1A to 1B: and (3) building a Box-Behnken model of the cross section area of the natural product and the muscle. 18 individual combinations and concentrations of the three natural products were studied using an incomplete factorial design. The 13 combinations are unique and the 5 are identical in order to determine biological variability in the system. FIG. 1A: average change in fiber cross-sectional area for each of the 18 different treatment groups after 14 days of overload. FIG. 1B: response surface plot of the relationship between CSA and the amount of epicatechin and epigallocatechin-3-gallate at constant levels of tripterine (500 μ g/kg/day).

Fig. 2A to 2H: and (3) verification of a Box-Behnken model of the relation between a natural product and the cross sectional area of muscle fibers. Body weight after 14 days of overload at different natural product levels (fig. 2A), (fig. 2B) heart weight/body weight, (fig. 2C) liver weight/body weight, (fig. 2D) muscle mass. FIG. 2E: the fiber cross-sectional area varies in distribution from left to right in the control, least effective, moderately effective, and most effective combinations of natural products. FIG. 2F: relationship between muscle fiber CSA and muscle mass 14 days after overload. Note that in this model, muscle mass increased by-80% before measurable changes in the average fiber CSA occurred. FIG. 2G: average fiber CSA as a function of overload and treatment. FIG. 2H: the relationship between the predicted change in fiber CSA from the Box-Behnken model and the actual measured change in fiber CSA after 14 days of overload. Data are mean ± SEM of n =5 animals per treatment group. * Indicating a significant difference from the control muscle.

Fig. 3A to 3C: overload and SIRT1 levels and acetylation of natural product treatments. FIG. 3A: levels of SIRT1 protein after overload and natural product treatment. FIG. 3B: overloaded and treated with natural products, acetylated at p53 at K382. Note that: the increase in p53 acetylation was greater with the natural product treatment. However, variability in each group precludes statistical significance. Finally, figure 3C shows the level of acetylation of lysine in the whole muscle homogenate. Data are mean ± SEM of n =5 animals per treatment group, with each dot shown. * Indicating a significant difference from the control muscle.

Fig. 4A to 4G: overloaded and natural product processed protein synthesis and ribosomal markers. FIG. 4A: protein synthesis estimated by SUnSET. Blot and quantitative data are shown. Ribosome mass was estimated by measuring (FIG. 4B) total RNA (80% of which is ribosomal RNA), (FIG. 4C) internal transcribed spacer 1 (ITS 1), and (FIG. 4D) 5 'external transcribed spacer (5' ETS). To obtain Akt-mTORC1 signaling, akt Ser473, (fig. 4F) S6K1 Thr389 and (fig. 4G) eEF2 phosphorylation were measured (fig. 4E). Data are mean ± SEM of n =5 animals per treatment group, with each dot shown. * Indicating a significant difference from the control muscle.

Fig. 5A to 5B: markers of protein turnover for overload and natural product processing. mRNA was measured for MuRF (FIG. 5A) and MaFBx (FIG. 5B). Data are mean ± SEM of n =5 animals per treatment group, with each dot shown. * Indicating a significant difference from the control muscle.

Fig. 6A to 6C: acetylation of overloaded and natural product treated ribosomal proteins and regulators. To obtain estimates of ribosome acetylation, representative proteins from the large (L13A) (fig. 6A) and small (S6) (fig. 6B) ribosomal subunits were blotted after immunoprecipitation with acetyl-lysine antibody. FIG. 6C: the level of S6K1 acetylation was also determined in the same manner. Note the opposite mode of the two measurements. Data are mean ± SEM of n =5 animals per treatment group, with each dot shown. * Representing a significant difference from the control muscle.

Detailed Description

1. Introduction part

The present application provides methods and compositions for enhancing load-induced muscle hypertrophy in a subject. In particular, the present application is based on the following surprising findings: certain natural products can be combined in a particular manner to produce novel combinations that do not occur in nature and that can be used to safely and effectively enhance muscle hypertrophy in a subject, thereby increasing the mass and other characteristics of skeletal muscle in the subject. The methods and compositions of the present application can result in a significant increase in muscle mass relative to muscle subjected to a load without receiving the composition. For example, addition of a combination of natural products of the present application on top of a standardized loading program can result in an increase in muscle mass that is at least 30% greater than the increase seen with loading alone. Without being bound by theory, it is believed that the natural products of the present application inhibit Sirtuin 1 protein (SIRT 1), resulting in increased acetylation of ribosomes and other proteins, enhanced ribosome function, and enhanced muscle growth.

In some embodiments, the composition comprises a combination of natural compounds including one or more catechins and tripterine or a derivative thereof. In a specific embodiment, the composition comprises celastrol, epicatechin mono-gallate and epigallocatechin mono-gallate.

The natural products described herein can be administered to a subject in any of a variety of ways. In a particular embodiment, the natural product is administered orally, for example as a nutritional supplement or food additive. In some embodiments, the nutritional supplement or food additive is a pill, tablet, capsule, liquid, powder, energy bar, protein bar, or gel. In particular embodiments, the natural products included in the compositions are certified as Generally Recognized As Safe (GRAS), meaning that they can be readily formulated for human consumption, for example, as food additives.

2. Definition of

As used herein, the following terms have the meanings assigned to them unless otherwise specified.

The terms "a", "an", or "the" as used herein include not only aspects having one member, but also aspects having more than one member. For example, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the agent" includes reference to one or more agents known to those skilled in the art, and so forth.

The terms "about" and "approximately" as used herein generally refer to an acceptable degree of error in the measured quantity given the nature or accuracy of the measurement. Typically, exemplary degrees of error are within 20% (%) of a given value or range of values, preferably within 10%, more preferably within 5%. Any reference to "about X" specifically means at least X, 0.8X, 0.81X, 0.82X, 0.83X, 0.84X, 0.85X, 0.86X, 0.87X, 0.88X, 0.89X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.11X, 1.12X, 1.13X, 1.14X, 1.15X, 1.16X, 1.17X, 1.18X, 1.19X, and 1.2X. Thus, "about X" is intended to teach and provide written descriptive support for claim limitations such as "0.98X".

The terms "expression" and "expressed" refer to the production of a transcription and/or translation product, such as a nucleic acid sequence encoding a protein (e.g., SIRT 1). In some embodiments, the term refers to the production of a transcription and/or translation product encoded by a gene (e.g., a human SIRT1 gene) or a portion thereof. The level of expression of a DNA molecule in a cell can be assessed based on the amount of the corresponding mRNA present within the cell or the amount of protein encoded by the DNA produced by the cell.

As used herein, "catechin" refers to a family of flavanols or flavan-3-ols and derivatives thereof. Catechins are members of the flavonoid family and occur naturally in, for example, cocoa, chocolate, tea and grapes. The catechins have two benzene rings and a dihydropyran heterocycle having a hydroxyl group at carbon 3. Catechins have four diastereoisomers, two of which are in the trans configuration (known as "catechins") and two of which are in the cis configuration (known as "epicatechins"). As used herein, the term "catechin" may generally refer to any type of catechin, including epicatechin, epigallocatechin, gallocatechin, and gallate derivatives of each of these. In some embodiments, the catechin included in the compositions of the present invention is epicatechin. As used herein, "epicatechin" refers to two diastereomers, namely (-) -epicatechin (see, e.g., pubChem CID 72276) or (+) -epicatechin (see, e.g., pubChem CID 182232) and derivatives thereof. For example, "epicatechin" may refer to epigallocatechin (see, e.g., pubChem CID 72277), flavan-3, 3',4', 5', 7-hexanol, i.e., (-) -epigallocatechin or (+) -epigallocatechin, and esters of epicatechin or epigallocatechin and gallic acid. For example, epigallocatechin gallate, epigallocatechin-3-gallate, epigallocatechin mono-gallate, epicatechin mono-gallate and the like. In particular embodiments, the catechin is epicatechin gallate (or epicatechin mono gallate) (see, e.g., pubChemCID107905; mol.wt.442.4 g/mol) and/or epigallocatechin gallate (or epigallocatechin mono gallate, or epigallocatechin-3-mono gallate) (see, e.g., pubChem CID65064; mol.wt.458.4 g/mol).

"Tripterine (celastrol)" as used herein refers to a pentacyclic triterpenoid compound originally isolated from Tripterygium wilfordii, having an exemplary structure as shown in PubChem CID 122724 (mol.wt.450.6 g/mol). Compositions of the invention also include a tripterine derivative, such as dihydrotripterine (see, e.g., pubChem CID 10411574), which is a compound synthesized by hydrogenation of tripterine.

"SIRT1" or "sirtuin-2 homolog 1" are members of class 1 of the sirtuin family of proteins, which are homologs of yeast Sir2 protein. SIRT1 is an NAD + dependent deacetylase and is activated in muscle in response to changes in cellular energy flux. SIRT1 can deacetylate proteins such as p53 and ribosomal S6 protein kinase (S6K 1). Information about the function, structure, localization, etc. of SIRT1 proteins can be found, inter alia, in UniProt Q96EB6 (SIR 1_ HUMAN); the SIRT1 gene corresponds, for example, to NCBI gene ID No. 23411.

By "SIRT1 inhibitor" is meant any agent, e.g., a natural product, such as erygiene or a catechin (e.g., epicatechin mono-gallate or epigallocatechin-3-mono-gallate), that is capable of inhibiting, reducing, decreasing, attenuating, eliminating, removing, slowing, or counteracting any aspect of SIRT1 expression, stability, or activity in any manner. A SIRT1 inhibitor can, for example, reduce any aspect of expression (e.g., transcription, RNA processing, RNA stability, or translation) of a gene encoding SIRT1 (e.g., a human SIRT1 gene) by, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, as compared to a control, e.g., in vitro or in vivo, in the absence of the inhibitor. Similarly, a SIRT1 inhibitor may, for example, reduce the activity, e.g., enzymatic activity, e.g., deacetylase activity on a substrate (such as p53 or S6K 1), of a SIRT1 enzyme by, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, compared to a control, e.g., in vitro or in vivo, in the absence of the inhibitor. In addition, a SIRT1 inhibitor may, for example, reduce the stability of a SIRT1 enzyme by, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, compared to a control, e.g., in vitro or in vivo, in the absence of the inhibitor. In particular embodiments, the SIRT1 inhibitor is a natural product, such as celastrol, a celastrol derivative, or a catechin or epicatechin, such as epicatechin mono-gallate or epigallocatechin-3-mono-gallate.

In the context of compounds, the term "derivative" includes, but is not limited to, amide, ether, ester, amino, carboxyl, acetyl and/or alcohol derivatives of a given compound.

The terms "administering," "administering," or "administering" refer to a method useful for enabling a medicament or composition (e.g., a compound described herein) to be delivered to a desired site of biological action. These methods include, but are not limited to, parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial, intravascular, intracardiac, intrathecal, intranasal, intradermal, intravitreal, etc.), transmucosal injection, oral administration, administration as a suppository, and topical administration. In particular embodiments of the present disclosure, compositions, e.g., combinations of SIRT1 inhibiting compounds, are natural products and are formulated, e.g., as a dietary or nutritional supplement or food additive for oral administration.

As used herein, "natural product" refers to compounds that occur in nature, i.e., compounds produced from living sources and derivatives thereof. The natural products used in the process of the invention may be isolated from natural sources, for example as extracts, or may be chemically synthesized (fully synthetic or semi-synthetic). The "natural product" used in the methods and compositions of the invention can be the natural product itself (e.g., tripterine) or a derivative thereof (e.g., dihydrotripterine, synthesized by hydrogenation of tripterine). The combination of natural products used in the methods and compositions of the present invention is novel and does not occur in nature.

Muscle "hypertrophy" refers to muscle growth occurring through an increase in the size of skeletal muscle cells in response to an increase in load, without an accompanying increase in the number of muscle fibers. Muscle hypertrophy may be detected by, for example, an increase in fiber cross-sectional area, an increase in muscle mass, an increase in muscle performance, or an increase in other parameters.

The term "treating" refers to any of the following: ameliorating one or more symptoms of the disease or disorder; preventing the manifestation of these symptoms before they occur; slowing or completely preventing the progression of the disease or disorder (e.g., a significantly longer period between recurrent episodes, slowing or preventing worsening of symptoms, etc.); enhancing the onset of remission; slowing irreversible damage caused in the progressive-chronic stage (in both the primary and secondary stages) of the disease or condition; delaying the start of the progression phase; or any combination thereof. In the context of the present disclosure, the methods and compositions of the invention may be used, for example, to increase muscle growth, strength, mass, or to treat the performance of a condition or disease, such as muscle atrophy caused by a condition or disease, such as post-operative recovery, restricted mobility, diabetes, cardiovascular disease, and cancer.

In some embodiments, the methods and compositions are used to enhance muscle growth, strength, quality, or function in healthy individuals, such as in the absence of muscle atrophy, e.g., in individuals for whom increased muscle mass is desired for aesthetic, athletic, fitness-related, health-related, or other reasons.

The term "effective amount" or "effective dose" or "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a compound (e.g., a SIRT1 inhibitor) sufficient to produce a beneficial or desired clinical or physiological effect. For example, in the present disclosure, a therapeutically effective amount or dose of a compound or natural product can be any amount or dose that increases or enhances one or more aspects of muscle function, mass, hypertrophy, strength, performance or other characteristic in a subject. An effective amount or dose may be based on factors per individual subject including, but not limited to, the age, size, physical health level, diet, genetic background, presence of any disease or disorder, route of administration, type or extent of any supplemental therapy used, etc. of the subject. In some embodiments, an effective amount of a compound or natural product as described herein can be initially estimated, for example, from cell culture or in vitro assays (e.g., by measuring SIRT1 inhibition) or animal models (e.g., by assessing muscle growth, function, quality, strength, etc.).

The terms "subject" and "individual" and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, rats, simians, humans, farm animals, or livestock for human consumption, such as pigs, cattle, and sheep, as well as sport animals and pets.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-or double-stranded form, and polymers thereof. Unless specifically limited, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences and as well as the sequence explicitly indicated.

"polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term as used herein includes amino acid chains of any length, including full length proteins, in which the amino acid residues are linked by covalent peptide bonds.

Detailed Description

The present application is based on the following surprising findings: specific combinations of natural products can promote skeletal muscle hypertrophy and thereby increase various characteristics of muscle, including mass, strength, durability, fiber CSA (cross-sectional area), performance, and function. The product may be administered by any route, including orally, for example as a nutritional supplement or food additive. In some embodiments, administration of the compound results in a decrease in SIRT1 level or activity, and/or an increase in acetylation of SIRT1 substrates such as p53 or ribosomal S6 protein kinase (S6K 1).

In some embodiments, the hypertrophic muscle is subjected to a load, e.g., by weight training or resistance training, which is concurrent with administration of the natural product. Although the load of skeletal muscle, even in the absence of the compounds of the present invention, generally results in hypertrophy and increases in, for example, muscle mass, fiber CSA, and strength, the extent of hypertrophy and increases in, for example, muscle mass, fiber CSA, and strength are significantly greater in the presence of the compounds of the present invention than in the absence of the compounds of the present invention. In some embodiments, the increase in hypertrophy, muscle mass, strength, or other characteristic in the presence of natural product and load is at least 10%, 20%, 30%, 40%, 50% or more greater than the increase seen in the presence of load but in the absence of natural product. In some embodiments, the loading is performed in the absence of aerobic exercise. In some embodiments, the natural product is administered in parallel with an amino acid or protein that promotes muscle growth, such as leucine, branched chain amino acids, or a protein with a high leucine content, such as whey protein. Such amino acids or proteins may be formulated with the natural product or separately and may be administered simultaneously with the product or according to a separate regimen. In some embodiments, caloric intake of a subject is increased concurrently with administration of a compound of the invention and muscle load.

Test subject

The subject may be any subject, e.g., a human or other mammal, in which it is desirable to increase muscle hypertrophy, growth, mass, strength, performance or function. In some embodiments, the subject is a human. In some embodiments, the subject is an adult. In some embodiments, the subject is a child. In some embodiments, the subject is a juvenile. In some embodiments, the subject is a female. In some embodiments, the subject is male.

In some embodiments, the subject has a disease or disorder associated with loss of muscle mass or function (e.g., muscle atrophy), such as diabetes, cardiovascular disease, restricted activity, cancer, cachexia, or post-operative recovery, and the natural product is administered to restore or increase the mass, function, fibrous CSA, or other characteristic of the atrophic muscle in the subject. In general, any disease or condition that involves loss of skeletal muscle mass or function, or that may benefit in any way from an increase in the mass or function of one or more skeletal muscles, may be treated using the methods and compositions of the present invention.

In some embodiments, the subject does not have a disease or disorder associated with loss of muscle mass or function, and does not have a muscle that is atrophied, but rather desires to increase muscle mass or function for another reason, such as increasing strength, improving coordination, enhancing motor performance, increasing bone density, improving metabolism, strengthening ligaments and tendons, reducing the risk of injury, or for aesthetic reasons.

In particular embodiments, the compositions of the invention are administered in conjunction with a loading of skeletal muscle (e.g., a loading of skeletal muscle in the absence of aerobic exercise). For example, the load may be performed by weight training (e.g., free weight), a weight lifting machine, a resistance band, or exercise against using the subject's weight. The load may be continuous or intermittent, high or low, with, for example, fewer or more repetitions per group, respectively. The load may be concentrated on one or a few muscles, or more generally on muscles of the whole body.

The methods and compositions of the present invention can affect any skeletal muscle, comprises a pectoralis complex, latissimus dorsi, femoris and subscapularis, brachialis, biceps, brachialis, quadriceps, forearm, flexor radialis, flexor cervicis, flexor ulnaris, flexor superficial, flexor deep, flexor crus, biceps brachii, adductor brachii, flexor biceps brachii, iliocoris lumbalis, rectus lumbalis, abdominus, rectus femoris, gluteus maximus, gluteus medius, hamstring, gastrocnemius, lateral crus, and gluteus medius quadriceps femoris, adductor longus, adductor brevis, adductor major, gastrocnemius medial, gastrocnemius lateral, soleus, tibialis posterior, tibialis anterior, flexor longus, flexor brevis, flexor hallucis longus, eye muscle, pharyngeal muscle, sphincter muscle, hand muscle, arm muscle, foot muscle, leg muscle, chest muscle, abdominal muscle, back muscle, hip muscle, shoulder muscle, head and neck muscle, etc.

In some embodiments, the composition results in a decrease in SIRT1 activity in a muscle of the subject. In some embodiments, the composition results in increased acetylation of one or more ribosomal proteins in the subject. In some embodiments, the composition does not substantially alter the weight of the subject or the weight of the heart or liver.

Assessment of SIRT1 levels

Any of a variety of methods may be used to assess the level or activity of SIRT1 in muscle, for example, when assessing the efficacy of a SIRT1 inhibitor or when assessing the level or activity of SIRT1 in a subject. For example, the level of SIRT1 may be assessed indirectly by examining transcription of a gene encoding SIRT1 (e.g., a SIRT1 gene), by examining the level of SIRT1 protein, by measuring SIRT1 enzyme activity, or by measuring, for example, acetylation of a SIRT1 substrate such as p 53.

In some embodiments, the methods involve measurement of SIRT1 enzyme activity, e.g., using standard methods, e.g., in the presence of SIRT1 and p53, incubating the candidate compound in an appropriate reaction buffer (e.g., containing excess nicotinamide adenine dinucleotide), and monitoring deacetylation by mobility shift analysis based on charge differences before and after electrophoretic separation of products, e.g., using a Reader such as Caliper EZ Reader (see, e.g., example 1).

In some embodiments, the methods involve detection of SIRT 1-encoding polynucleotide (e.g., mRNA) expression, which can be analyzed using conventional techniques, such as RT-PCR, real-time RT-PCR, semi-quantitative RT-PCR, quantitative polymerase chain reaction (qPCR), quantitative RT-PCR (qRT-PCR), multiple-branched DNA (bDNA) assays, microarray hybridization, or sequence analysis (e.g., RNA sequencing ("RNA-Seq")). Methods for quantifying polynucleotide expression are described, for example, in Fassbinder-Orth, integrated and Comparative Biology,2014, 54; thellin et al, biotechnology Advances,2009, 27; and Zheng et al, clinical Chemistry,2006,52 (doi: 10/1373/clinchem.2005.065078). In some embodiments, real-time or quantitative PCR or RT-PCR is used to measure the level of a polynucleotide (e.g., mRNA) in a biological sample, see, e.g., nolan et al, nat. Protoc,2006,1, 1559-1582; wong et al, bioTechniques,2005, 39. Quantitative PCR and RT-PCR assays for measuring gene expression are also commercially available (e.g.,

Gene Expression Assays,ThermoFisher Scientific)。

in some embodiments, the methods include detecting SIRT1 protein expression or stability, for example, using conventional techniques known to those skilled in the art, such as immunoassays, two-dimensional gel electrophoresis, and quantitative mass spectrometry. Protein quantification techniques are generally described in "Strategies for protein quantification," Principles of Proteomics, 2 nd edition, ed by r.twyman, garland science,2013. In some embodiments, protein expression or stability is detected by an immunoassay, such as, but not limited to, an Enzyme Immunoassay (EIA), such as an enzyme-multiplied immunoassay technique (EMIT), an enzyme-linked immunosorbent assay (ELISA), an IgM antibody capture ELISA (MAC ELISA), and a particulate enzyme immunoassay (MEIA); capillary Electrophoresis Immunoassay (CEIA); radioimmunoassay (RIA); immunoradiometric assay (IRMA); immunofluorescence (IF); fluorescence Polarization Immunoassay (FPIA); and chemiluminescence assay (CL). Such immunoassays can be automated, if desired. Immunoassays can also be used in conjunction with laser-induced fluorescence (see, e.g., schmalzing et al, electrophoresis,18 2184-93 (1997); bao, j. Chromatographic.b. Biomed. Sci., 699.

The natural product may enhance any of a number of characteristics of muscle, including mass, strength, fiber cross-sectional area (CSA), protein content, fiber volume, muscle efficiency, performance, and the like. Muscle growth and performance can be measured using any of a variety of methods, such as imaging (e.g., X-ray, MRI, CT, ultrasound), by molecular or cellular analysis, such as a muscle biopsy taken from a biopsy of the subject, or by any functional test, such as a grip test, walking speed, muscle strength test, resistance test, treadmill, or other functional test.

Compound (I)

The present application is based on the following surprising findings: particular combinations of natural products may promote hypertrophy in skeletal muscle (e.g., skeletal muscle undergoing stress). The combinations described herein comprise one or more catechins and tripterine or a derivative thereof. In some embodiments, the one or more catechins and celastrol or a derivative thereof inhibits SIRT1, e.g., results in at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of the expression, stability, or activity of SIRT1, relative to a control level, e.g., in the absence of an inhibitor, in vivo or in vitro. In one embodiment, SIRT1 activity is assessed in vitro using p53 as a substrate, for example by examining the acetylation of lysine 382, for example by measuring the mobility change caused by SIRT1 deacetylase activity, for example as described in example 1.

In some embodiments, the one or more catechins and tripterine or derivatives thereof cause an increase in one or more properties of one or more muscles in the subject, e.g., growth, mass, strength, performance, fiber volume, fiber cross-sectional area, e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, as compared to a control level, e.g., the level in the absence of one or more catechins and tripterine or tripterine derivatives.

In some embodiments, the one or more catechins included in the combination are epicatechins. In a particular embodiment, the one or more epicatechin included in the combination is epicatechin mono-gallate and epigallocatechin-3-mono-gallate. The combination may comprise any of a variety of amounts of natural products. For example, the combination can be formulated and administered such that the subject receives about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5. 9. 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mg/kg/day or more of any compound. In some embodiments, the combination is formulated and administered such that the subject receives about 0.7-1.3 mg/kg/day of epicatechin mono gallate. In some embodiments, the combination is formulated and administered such that the subject receives about 0.7 mg/kg/day of epicatechin mono gallate. In some embodiments, the combination is formulated and administered such that the subject receives about 6-20 mg/kg/day of epigallocatechin-3-mono-gallate. In some embodiments, the combination is formulated and administered such that the subject receives about 20 mg/kg/day of epigallocatechin-3-mono gallate.

In some embodiments, the combination comprises tripterine or a tripterine derivative, such as dihydrotripterine. In some embodiments, the combination is formulated and administered such that the subject receives about 0.2-0.5 mg/kg/day of celastrol or celastrol derivative. In some embodiments, the combination is formulated and administered such that the subject receives about 0.5 mg/kg/day of tripterine or a tripterine derivative. In some embodiments, the combination is formulated and administered such that the subject receives about 0.7-1.3 mg/kg/day of epicatechin mono-gallate, about 6-20 mg/kg/day of epigallocatechin-3-mono-gallate, and about 0.2-0.5 mg/kg/day of tripterine. In a specific embodiment, the combination is formulated and administered such that the subject receives about 0.7 mg/kg/day of epicatechin mono-gallate, about 20 mg/kg/day of epigallocatechin-3-mono-gallate, and about 0.5 mg/kg/day of tripterine. In some embodiments, the combination is formulated (and/or administered) such that the relative weight ratio of each of epicatechin mono-gallate, epigallocatechin-3-mono-gallate and tripterine (and/or received by the subject, e.g., each administration or each day) in the composition is about 0.7. In some embodiments, the combination is formulated (and/or administered) such that the relative molar ratio of each of epicatechin mono-gallate, epigallocatechin-3-mono-gallate and tripterine (and/or received by the subject, e.g., each administration or each day) in the composition is about 1.6.

In some embodiments, celastrol or a celastrol derivative is administered alone or in combination with one or more other compounds other than catechins. In some embodiments, the celastrol or celastrol derivative, alone or in combination with one or more non-catechin compounds, results in at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more reduction in expression, stability, or activity of SIRT1 relative to a control level, e.g., in the absence of celastrol, celastrol derivative, or a combination comprising celastrol, in vivo, or in vitro. In some embodiments, celastrol or celastrol derivatives alone in combination with one or more non-catechin compounds results in an increase in one or more properties of one or more muscles in a subject, such as growth, mass, strength, performance, fiber volume, fiber cross-sectional area, compared to a control level, for example, a level of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the absence of celastrol, celastrol derivatives, or a combination comprising celastrol.

When administered alone or in combination with one or more additional non-catechin compounds, the tripterine or tripterine derivative may be formulated and administered in any range of amounts, e.g., such that the subject receives about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6.1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mg/kg/day or more of any compound. In some embodiments, the tripterine or tripterine derivative is formulated and administered such that the subject receives about 0.2-0.5 mg/kg/day of tripterine or tripterine derivative. In some embodiments, the celastrol or celastrol derivative is formulated and administered such that the subject receives about 0.5 mg/kg/day of celastrol or celastrol derivative.

The natural products described herein can be obtained from a variety of sources, including from natural sources, by chemical synthesis, and from chemical suppliers. For example, epicatechin mono gallate can be isolated, for example, from green tea, grape, or obtained, for example, from Aurora fine chemicals, sigma-Aldrich, combi-Blocks, chemShuttle, and the like. Epigallocatechin mono gallate may be isolated, for example, from green or black tea, or obtained, for example, from Sigma-Aldrich, combi-Blocks, king Scientific, and the like. Can be isolated from root extracts such as Tripterygium wilfordii and Celastrus regelii or obtained from, for example, aurum Pharmatech, achemtek, chemshuttle, VWR, etc. Dihydrotripterine can be synthesized, for example, by hydrogenation of tripterine, or can be obtained, for example, from Abovchem, achemtek, aurumPharmatech, sigma-Aldrich, and the like.

Formulation and administration

The compounds disclosed herein can be formulated and administered in any of a variety of ways. In some embodiments, the compounds are formulated as pharmaceutical compositions, i.e., comprising a pharmaceutically acceptable carrier. In certain aspects, the pharmaceutically acceptable carrier is determined in part by the particular composition being administered and the particular method used to administer the composition. Thus, there are a variety of suitable formulations of Pharmaceutical compositions of the compounds of the present invention (see, e.g., remington's Pharmaceutical Sciences, 18 th edition, mack Publishing co., easton, PA (1990)).

As used herein, "pharmaceutically acceptable carrier" includes any standard pharmaceutically acceptable carrier known to those of ordinary skill in the art for formulating pharmaceutical compositions. Thus, the compounds themselves, for example, as pharmaceutically acceptable salts or as conjugates, may be prepared as formulations in pharmaceutically acceptable diluents; for example, saline, phosphate Buffered Saline (PBS), aqueous ethanol, or solutions of glucose, mannitol, dextran, propylene glycol, oils (e.g., vegetable oils, animal oils, synthetic oils, etc.), microcrystalline cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, magnesium stearate, calcium phosphate, gelatin, polysorbate 80, etc., or as a solid formulation in a suitable excipient.

The pharmaceutical compositions will typically further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextran), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylated hydroxyanisole, and the like), bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of the recipient, suspending agents, thickening agents, preservatives, flavoring agents, sweetening agents and coloring compounds.

The pharmaceutical compositions described herein are administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The amount to be administered depends on various factors including, for example, the age, weight, physical activity and diet of the individual, any condition or disease to be treated, and the stage or severity of any underlying condition or disease. In certain embodiments, the size of the dose may also be determined by the presence, nature and extent of any adverse side effects that accompany the administration of the therapeutic agent in a particular individual.

It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, genetic characteristics, general health, sex, diet, mode and time of administration, rate of excretion, drug combination. The presence and severity of any particular condition, as well as any other potential therapies administered.

In certain embodiments, the dosage of the compounds may take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, and the like, preferably in unit dosage forms suitable for simple administration of precise dosages.

As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for humans and other mammals, each unit containing a predetermined quantity of therapeutic agent calculated to produce the desired seizure, tolerability and/or therapeutic effect, in association with a suitable pharmaceutical excipient (e.g., an ampoule). In addition, more concentrated dosage forms can be prepared, from which more dilute unit dosage forms can then be prepared. Thus, a more concentrated dosage form will substantially contain more than, e.g., at least 1, 2, 3, 4,5, 6, 7,8, 9, 10-fold or more, of the therapeutic compound.

Methods for preparing such dosage forms are known to those skilled in the art (see, e.g., remington's Pharmaceutical Sciences, supra). The dosage form typically includes conventional pharmaceutical carriers or excipients, and may additionally include other agents, carriers, adjuvants, diluents, tissue penetration enhancers, solubilizers, and the like. Suitable excipients can be tailored to the particular dosage form and route of administration by methods well known in the art (see, e.g., remington's Pharmaceutical Sciences, supra).

Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopol, e.g., carbopol 941, carbopol 980, carbopol 981, and the like. The dosage form may additionally include lubricants, such as talc, magnesium stearate, and mineral oil; a wetting agent; an emulsifier; a suspending agent; preservatives, such as methyl-, ethyl-, and propyl-hydroxy-benzoate (i.e., parabens); pH adjusters such as inorganic and organic acids and bases; a sweetener; and a flavoring agent. The dosage form may also comprise biodegradable polymer beads, dextran and cyclodextrin inclusion complexes.

In specific embodiments, the compounds are formulated for oral, buccal, or sublingual administration. For example, a therapeutically effective dose can be in the form of tablets, capsules, pills, pellets, soft capsules, gums, emulsions, suspensions, solutions, syrups, elixirs, pastes, gels, granules, gums, liquids, powders, fast dissolving tablets, effervescent formulations, sachets, semi-solids, sprays, lozenges, powders, tinctures, and sustained release formulations. Suitable excipients for administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.

In particular embodiments, the natural product is formulated as a nutritional supplement or food additive, e.g., as a pill, tablet, capsule, liquid, powder, energy bar, protein bar, gum, chocolate, candy, mint, and the like, optionally containing other elements, e.g., sweeteners, flavors, colorants, proteins, amino acids such as leucine or other branched chain amino acids, and the like.

Compositions for oral administration may optionally comprise, for example, carrier materials such as corn starch, acacia, gelatin, malt, tragacanth, microcrystalline cellulose, kaolin, dicalcium phosphate, calcium carbonate, sodium chloride, alginic acid, lactose, glucose or sucrose, in addition to the natural products described herein; disintegrating agents, such as microcrystalline cellulose or alginic acid; binders, such as acacia, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, or hydroxypropylmethylcellulose; and/or lubricants, such as magnesium stearate, stearic acid, silicone fluids, talc, oils, waxes, colloidal silica, and the like.

The natural product may also be provided in lyophilized form. Such dosage forms may include a buffer, such as bicarbonate, for reconstitution prior to administration, or the buffer may be included in a lyophilized dosage form for reconstitution (e.g., with water). The lyophilized dosage form may also contain a suitable vasoconstrictor, such as epinephrine. The lyophilized dosage form may be provided in a syringe, optionally packaged in combination with a buffer for reconstitution, such that the reconstituted dosage form may be immediately administered to an individual.

In some embodiments, additional compounds or drugs may be co-administered to the subject. Such compounds or drugs may be co-administered to alleviate signs or symptoms of the disease being treated, to reduce side effects caused by induction of immune responses, and the like. In some embodiments, for example, the natural product is administered with an amino acid that promotes muscle growth, such as leucine or a branched chain amino acid, or with a leucine-rich protein, and/or with any other compound intended to enhance muscle mass, strength, or function, such as another SIRT1 inhibitor.

The compounds of the invention may be administered locally or systemically in a subject. In some embodiments, the compound can be administered, e.g., intraperitoneally, intramuscularly, intraarterially, orally, intravenously, intracranially, intrathecally, intraspinally, intralesionally, intranasally, subcutaneously, intracerebroventricularly, topically, transdermally, sublingually, buccally, and/or by inhalation. In particular embodiments, the compound is administered orally, e.g., as a food supplement.

In some embodiments, the compound is administered to the subject once. In other cases, the compound is administered at one time point and again at a second time point. In other instances, the compound is administered to the subject repeatedly (e.g., once or twice daily) in intermittent doses over a limited period of time (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or longer). In some cases, the time between administration of the compounds is about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or more. In other embodiments, the compound is administered continuously or chronically over a desired period of time. For example, the compound may be administered such that the amount or level of the compound is substantially constant over a selected period of time. In some embodiments, the compound is administered over an extended period of time, e.g., several months or more, e.g., concurrently with a weight training program of indeterminate duration.

The compounds may be administered to a subject by methods commonly used in the art. The amount of compound introduced will take into account factors such as the sex, age, weight, presence or absence of a disease or disorder, presence or absence of muscle atrophy, the specific goals and motivation of the subject to increase muscle mass, strength, or function, and the amount required to produce the desired result. Typically, for administration of a compound for therapeutic or other purposes, the compound is administered in an "effective dose" or "therapeutically effective dose". An "effective amount" or "effective dose" is an amount sufficient to produce a desired physiological effect or to achieve a desired result, including for treating a condition or disease, e.g., to reduce or eliminate one or more symptoms or manifestations of a condition or disease, and for improving muscle mass in the absence of a disease or condition.

Any number of muscles of the body may be hypertrophied by the presence of the compounds described herein, e.g., the biceps; the triceps muscle; the radial muscle of the arm; arm muscles (arm dorsal muscles); superficial flexor carpi; a deltoid muscle; biceps femoris, gracilis, semitendinosus and semimembranosus of the retrofemoral muscle group; rectus femoris, vastus lateralis, vastus medialis, and vastus intermedius of the quadriceps femoris; gastrocnemius (lateral and medial) of the lower leg, tibialis anterior and soleus muscles; the major and minor pectoralis muscles of the chest; latissimus dorsi of the upper back; rhomboid (large, small); trapezius muscles spanning the neck, shoulders and back; abdominal rectus muscle; gluteus maximus, gluteus medius, and gluteus minimus of the buttocks; the hand muscles; a sphincter; eye muscles; and pharyngeal muscles.

4. Reagent kit

Other embodiments of the compositions described herein are kits comprising a compound described herein. Kits typically comprise a container, which may be formed of a variety of materials such as glass or plastic, and may include, for example, bottles, vials, syringes, and test tubes. The label typically accompanies the kit and includes any written or recorded material, which can be in electronic or computer readable form, providing instructions or other information for using the kit contents.

In some embodiments, the kit comprises one or more agents for promoting muscle growth or hypertrophy. In some embodiments, the kit comprises one or more catechins and tripterine or a derivative thereof. In some embodiments, the kit comprises two catechins and tripterine or a derivative thereof. In some embodiments, the two catechins are epicatechins. In some embodiments, the epicatechin is epicatechin-3-mono-gallate and epigallocatechin mono-gallate. In some embodiments, the kit further comprises one or more additional agents, such as one or more muscle growth promoting amino acids or proteins, such as leucine or branched chain amino acids, or leucine rich proteins, such as whey proteins.

In some embodiments, the kit can also include instructional materials (e.g., instructions for using the kit for enhancing the quality, strength, or function in an atrophic or non-atrophic muscle) containing instructions (i.e., a protocol) for performing the methods of the invention. Although the instructional materials typically comprise written or printed material, they are not limited thereto. The present disclosure contemplates any medium that is capable of storing such instructions and communicating them to an end user. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tape, cassettes, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

5. Examples of the invention

Example 1 use of Natural product SIRT1 inhibitors to increase muscle hypertrophy

Introduction to the design reside in

To determine whether SIRT1 inhibits muscle growth, we group previously removed gastrocnemius and soleus muscles in wild type and muscle-specific SIRT1 knockout mice (mKO) and determined compensatory growth in the plantaris muscle (PLN). In SIRT1 mKO mice, muscle mass increased by 113% or more during 2 weeks of overload compared to Wild Type (WT) mice. Furthermore, in mice overexpressing SIRT1 there was a small but significant damage to muscle growth compared to WT mice. These data support the hypothesis that SIRT1 inhibits overload-induced muscle growth.

The current work targets are twofold: first, we sought to identify natural products capable of inhibiting SIRT 1; secondly, we sought to determine whether these natural products, when combined in an optimal manner, could increase muscle hypertrophy. In summary, our hypothesis is that we can find a new nutritional supplement that can increase the effect of overload on the cross-sectional area of muscle fibers.

Materials and methods

SIRT1 inhibitor screening

NatProd Collection library (Microsource Discovery System, inc. Gaylordsville, CT) was screened in 10 source plates at two doses (final 50. Mu.M and 5. Mu.M in the reaction mixture, two doses each). The inhibitory activity of the compounds was assessed against 2 ng/. Mu.l of purified human SIRT1 using 3. Mu.M p53 as substrate. The assay is based on a mobility shift assay of the charge difference before and after electrophoretic separation of the product from a fluorescently labeled substrate read using a Caliper EZ Reader (Perkin Elmer, boston, MA). All reactions occurred in the presence of excess nicotinamide adenine dinucleotide and the known SIRT1 inhibitor suramin (suramin) was used as a control.

Box-Behnken model generation

The three inhibitors identified in the natural product screen were selected based on previous use in humans and complementary chemical structures. Then use

The software used these inhibitors to generate the incomplete multi-factorial design Box-Behnken model. A three-factor design with one central point required 13 animals. An additional 5 animals received a central dose of all three natural products to determine biological variability.

Synergist ablation

All Animal procedures were approved by the Institutional Animal Care and Use Committee (Institutional Animal Care and Use Committee) at davis, university, california. 18 rats were used for DOE and validation experiments. Animals were anesthetized with 2.5% isoflurane, shaved and prepared for sterile surgery. The entire calcaneus and the lower half of the gastrocnemius muscle were removed at the achilles tendon leaving the metatarsals (PLN) intact. The upper fascia and skin were sutured closed and the animal was moved to a temperature-regulated area for recovery. The left leg was used as contralateral control. Animals were monitored daily to ensure that they returned to normal activity and were not subjected to any stress from the procedure. Animals were fed daily according to their respective treatment groups prior to light exposure.

Muscle harvesting

On day 14 post-treatment, animals were anesthetized and overloaded and contralateral PLN muscle, heart and liver were collected. After removal, the PLN muscles were trimmed conservatively, weighed, then pinned at rest length on cork, snap frozen in liquid nitrogen cooled isopentane, and stored at-80 ℃.

Histology

PLN muscles were closed on cork using OTC in a cryostat and 10 μm sections were mounted on glass slides for CSA quantification. Slides were prepared for histological analysis by blocking in 5% Normal Goat Serum (NGS) in PBST w/1% tween and incubating overnight at 4 ℃ in primary antibodies to type I, IIa, IIb fibers and/or laminin. The next day, slides were washed with PBST w/0.1% tween and incubated in HRP-conjugated secondary antibody for 60 minutes, washed again, and mounted using prolong gold (no Dapi). Four random images of each respective muscle section were taken using Fiji for CSA quantification.

Verification experiment

Following booster ablation, animals were randomized into one of four treatment groups, control (n = 3), minimal (n = 5), moderate (n = 5), and optimal (n = 5). The control group received Phosphate Buffered Saline (PBS), while the minimal, medium and best groups received 3 SIRT1 inhibitors (as a single mixture dissolved in PBS) in different combinations and concentrations. All treatments were administered by oral gavage prior to light exposure over 14 days.

mRNA isolation, reverse transcription and qPCR

After blocking, the PLN muscle was broken into powder with a hammer and pestle. Total RNA was extracted from powdered muscle tissue using RNAzol according to the manufacturer's protocol. RNA was quantified by absorbance (Biotek, winooski, VT) using a Biotek Epoch Microplate Reader. 1.5. Mu.g of total RNA was converted to cDNA using MultiScribe Reverse Transcriptase and oligo (DT) primers. cDNA was diluted 1. qPCR was performed using CFX384 Touch Real-Time PCR Detection System (Bio-Rad, hercules, calif.) with Quantified Mastermix and Bio-Rad Sybr Green Mix solutions and Bio-Rad 384-well PCR plates. The PCR reaction was performed according to the manufacturer's instructions with the following primers:

rITS-1(fwd-TCCGTTTTCTCGCTCTTCCC-；

rev-CCGGAGAGATCACGTACCAC-),

r5E1TS(fwd-ACGCACGCCTTCCCAGAGG-；

rev-CGCGTCTCGCCTGGTCTTG-)。

gene expression was calculated using the delta delta delta threshold cycle method (Livak and schmitgen, 2001) and GAPDH was used as the housekeeping gene.

Tissue homogenates and western blots

Two sample scoops of powder were incubated in 250. Mu.L of sucrose lysis buffer (1M Tris, pH7.5,1M sucrose, 1mM EDTA,1mM EGTA,1% Triton X-100 and protease inhibitor complex). The solution was placed on a shaker at 4 ℃ for 60 minutes, rotated at 8,000g for 10 minutes, and the supernatant was transferred to a new Eppendorf tube, and then the protein concentration was determined using a DC protein assay (Bio-Rad, hercules, calif.). 750 μ g of protein was diluted in 4X Laemmli Sample Buffer (LSB) and boiled for 5 minutes. A10. Mu.L sample of protein was loaded onto Criterion TGX Stain-Free Precast Gel and run at a constant voltage of 200V for 45 minutes. The proteins were then transferred to Immobilon-P PVDF membrane, activated in methanol and normalized at 100V constant voltage for 60 min in transfer buffer. Membranes were blocked in 1% Fish Skin Gelatin (FSG) in TBST (Tris-buffered saline w/0.1% tween) and incubated overnight at 4 ℃ with the appropriate primary antibody diluted 1,000 in TBST or FSG. The next day, the membranes were washed with TBST for 5 minutes three times and the secondary antibody conjugated with peroxidase was incubated continuously in 0.5% non fat mill TBST solution at 1. Bound antibody was detected using a chemiluminescent HRP substrate detection solution (Millipore, watford, UK). Imaging and band quantification were performed using a BioRad assay.

Immunoprecipitation

Muscle powder was homogenized and Protein quantified as described above, and 500 μ G of Protein was placed in a tube containing 25 μ L of antibody-loaded Protein G-Dyna Beads, which were aliquoted in Eppendorf tubes, and immunoprecipitation was prepared using the guidance protocol (Thermo Scientific, protein G-Dyna Beads). The antibody for pulldown was used at a concentration of 1.6 μ L of the sample was loaded per well into Criterion TGX Stain-Free Precast Gel and performed using the Western blotting protocol described above.

Antibodies

Primary antibodies for western blotting and immunoprecipitation were diluted to a concentration of 1. Antibodies are from Cell Signaling Technology (Danvers, MA, united States) -total eEF2 (CS-2332S), P-53 (CS-2524S), phosphor-eEF 2 (CS-2331S), SIRT1 (CS-947S), ac-Lys (CS 9441S), phosphor-S6 (CS-5364S), ac-P53 (CS-252S), P-AKT (Ser 473) (CS-4060S), cytochrome-C (CS-4280S); santa Cruz Biotechnology (Santa Cruz, CA, united States) -rps6 (SC-13007), rpL13a (SC-390131), dystrophin (SC-465954); abcam (Cambridge, UK) -Total OxPhos (ab 110413); and Millipore-puromycin (MABE 343).

Statistics

All data were analyzed using GraphPad Prism Software (GraphPad Software, inc., la Jolla, CA). Tukey post hoc analysis is used to determine differences when there is an interaction. Statistical significance was set at p <0.05. All data are expressed as mean ± Standard Error Mean (SEM).

Results

SIRT1 inhibitor screening

High throughput screening of 800 natural products identified 45 compounds that inhibited SIRT1>65% at a concentration of 50. Mu.M. Of these, many compounds showed dose-dependent effects on SIRT1, with 35 compounds showing at least 20% inhibition at 5 μ M. Of the 35 inhibitors identified, 3 have been widely used in human food (table 1) and are from 3 different chemical classes (one of quinone-methide, polyphenol and flavonoid). These compounds (tripterine, epigallocatechin-3-mono-gallate and epicatechin mono-gallate) were selected for further study with the aim of ameliorating muscle hypertrophy in mammals.

Doe model generation

The three compounds described above were input into a Box-Behnken incomplete factorial design to quickly assess any interaction between the different products. 13 rats received different combinations of the three products (0-2 mg/kg/day epicatechin; 0-10 mg/kg/day epigallocatechin-3-gallate; and 0-500 μ g/kg/day celastrol), while 5 controls received intermediate amounts of each product to determine biological variability. Overloading for 14 days resulted in a change in muscle fiber cross-sectional area that varied from-4.25% to 115.8% depending on treatment (figure 1), with the control averaging 66.8 ± 6.98%. From these data, the response surface plots indicate that epigallocatechin-3-gallate can modulate the effects of the other two products and generate a model that predicts the optimal combination and concentration of each product (FIG. 1B).

Model validation

To validate the model, a separate group of rats was subjected to booster ablations and then gavaged daily with saline controls or combinations of predicted best, least effective, or three predicted products to produce an increase in fiber CSA between the other two groups. The dose of each product per group is summarized in table 2. After 14 days of overloading and treatment, animals were sacrificed and body, heart, liver and muscle weights were determined (fig. 2). There were no statistical differences between the untreated and treated groups for body, heart or liver mass, indicating that treatment did not result in any acute toxicity. Both the medium and best groups showed a significant increase in muscle mass relative to control-treated rats. Analysis of muscle fiber CSA showed that the control leg showed a similar distribution of fiber CSA independent of treatment. In the best group, the right shift of the overloaded fiber CSA was the greatest. Mean fiber CSA for all groups of SHAM legs was 1847. + -. 114.6, 1945. + -. 132.5, 1883. + -. 114.6 and 1730. + -. 60.0 μm for the control, minimal, medium and optimal groups, respectively ² Whereas, for each group, the overload legs showed averages 1901 + -108.3, 2348.1 + -172.8, 2306.5 + -119.7 and 2800 + -145.9 μm ² (FIG. 2G). To test the Box-Behnken modelThe predicted change in fiber CSA was plotted against the measurements for each group. The resulting line had an r of 0.9586 ² Values, confirming the ability of the model to predict changes in muscle hypertrophy (fig. 2H).

SIRT1 levels and Activity

Since the treatment inhibited SIRT1, the level of SIRT1 and its enzymatic activity (p 53 acetylation) were determined (fig. 2). As previously described, SIRT1 levels significantly increased (-2-fold) in the control group after overload, and SIRT1 levels were even higher in the control and overloaded limbs after treatment with SIRT1 inhibitors. As a measure of SIRT1 activity, we determined the level of p53 acetylation at lysine 382. As other SIRT1 inhibitors have been reported (16), overloading in conjunction with treatment with natural product mixtures increases p53 acetylation at that residue; however, there was no difference in p53 acetylation at different doses. Finally, to determine if the natural product mix altered total protein acetylation, total acetylated proteins were measured and there were no statistical differences in total acetylated proteins with any treatment at the two week time point.

Protein synthesis reaction

To understand the mechanism by which the natural product increases CSA in muscle fibers, the rate of protein synthesis was determined by SuNSeT. Even though there was a trend of increasing baseline protein synthesis with the natural product mix, the increase in protein synthesis with overload was similar in all treatment groups (fig. 4A). Since ribosome mass is thought to control protein synthesis in extreme states (e.g., during overload), we next determined the rate of total RNA and ribosome biogenesis. Contrary to our hypothesis, total RNA tended to decrease from control to optimal treatment. Furthermore, when the rate of ribosomal RNA synthesis was determined by measuring the expression of the internal transcribed spacer 1 (ITS 1) and the 5' external transcribed spacer (5 ' ets), expression of these markers of ribosomal biogenesis was reduced from control to minimal to moderate to optimal, with 5' ets values significantly lower than control-treated muscles (fig. 4C to fig. 4D). To determine whether the increase in growth in the natural product group is a result of increased Akt-mTORC1 signaling, phosphorylation of Akt, S6K1, and eEF2 were determined. There was a trend of increased Akt phosphorylation with overload and decreased natural product (fig. 4E); however, none of these effects achieved significance. As previously described, S6K1 phosphorylation was higher in the overloaded leg (fig. 4F). Contrary to expectations, there was a trend towards a decrease in overload-induced S6K1 phosphorylation from control towards optimal natural product combinations; however, the activity of S6K1 (measured by eEF2 phosphorylation) did not differ in any of the overload groups (fig. 4G).

Protein turnover/degradation markers

Since the natural product had no effect on protein synthesis, markers of protein conversion were rapidly measured by measuring the expression of MuRF and MafBx. As previously described, muRF and MafBx expression increased with overload and was not affected by natural product handling (fig. 5).

Acetylation of ribosomal proteins

Ribosomal proteins are regulated by acetylation. Since SIRT1 is a deacetylase, acetylation of proteins representing small and large ribosomal subunits was determined after immunoprecipitation. With optimization of the natural product mixture, there was an increasing tendency for acetylation of ribosomal proteins (fig. 6A to 6B). In contrast, with optimization of the natural product mixture, S6K1 acetylation tended to decrease (fig. 6C).

Discussion of the preferred embodiments

Here, we show that several natural products have the ability to inhibit SIRT1 in an in vitro activity assay. Combining three of these natural products, which are generally considered to be safe (GRAS, the food and drug administration states that the chemicals added to food are considered by experts to be safe, thus obviating the requirement of federal food, drug and cosmetic act for resistance to food additives), in appropriate amounts results in a significant increase in muscle fiber hypertrophy after 14 days of overload. The significant increase in muscle fiber CSA was not the result of increased ribosome mass. Indeed, the best group showed significantly less 5' ets, with lower ITS1 levels and a strong trend of total RNA, indicating less ribosomes. Acetylation of ribosomal proteins tends to increase, indicating that the increase in myofibrillar proteins is likely the result of increased efficiency of the ribosomes rather than increased capacity. Importantly, the natural product mixture did not change body weight or heart and liver weight, indicating that it has limited toxicity and could be used to grow or maintain muscle mass.

We have previously identified SIRT1 as one of a series of molecular breaks that limit muscle growth in response to extreme stimuli (e.g., potentiator ablation). We hypothesized that activation of SIRT1 would lead to deacetylation of TAF68, a component of SL-1 transcription factor that drives 47S rRNA expression. Deacetylation of TAF68 has previously been shown to inhibit rRNA transcription and thus ribosome mass. Since ribosome mass is thought to limit growth after potentiator ablation (8, 11), we hypothesize that blocking SIRT1 will reduce TAF68 acetylation, increase rRNA expression, increase the ability of protein synthesis, and allow greater skeletal muscle hypertrophy in response to overload. Consistent with this hypothesis, we have previously shown that increased knockdown of SIRT1 and decreased overexpression of SIRT1 is responsive to overloaded muscle hypertrophy. In addition, pharmacological inhibitors of SIRT1 may increase load-induced muscle hypertrophy, suggesting that acute treatment with SIRT1 inhibitors may increase muscle hypertrophy in genetically normal animals. Using these data, we sought to determine whether SIRT1 could be inhibited by the natural product and produced the same improvement in growth.

Using NatProd Collection, which includes 800 pure natural products and their derivatives from plant, animal and microbial sources, we identified 45 compounds that inhibit SIRT1 activity at p53 greater than 65% at a concentration of 50 μ M, and 35 compounds that inhibit SIRT1 by at least 20% at 5 μ M. This represents a unique list of compounds, many of which are polyphenols, including quinones and flavonoids that inhibit SIRT1. The fact that most compounds that inhibit SIRT1 are polyphenols suggests that modulation of SIRT1 may be one reason that polyphenols have a significant impact on human health and disease prevention (13). We chose to focus on three of these polyphenols, epicatechin, epigallocatechin-3-gallate and celastrol, because they have a history of use in human medical trials without complications and have different chemical structures, which may imply different degrees of digestion, absorption, delivery and activity in the muscle after ingestion.

Use ofWithout complete factorial design, we treated animals with different concentrations and combinations of natural products to establish a model of how each natural product contributes to the increase in muscle CSA after overload. The response surface plot was used to determine the relative importance of each component and its interaction with other natural products in the mixture (FIG. 1B). To validate this model, we selected three different natural product combinations based on their predicted effect on muscle fiber CSA after overload. A group of independent rats (n =5 per treatment) received booster ablations followed by daily gavages containing one of the three combinations of natural products or placebo controls. Model prediction of fiber CSA increase is proportional to measured change in CSA (r) ² = 0.9586) indicates that the model is effective and that the predicted combination of epicatechin, epigallocatechin-3-gallate and tripterine is optimal for muscle hypertrophy.

The optimal combination of epicatechin, epigallocatechin-3-gallate and tripterine increased muscle fiber CSA by 61.5%, compared to-4% in the control group by 61.5%; thus, the increase in CSA in the best group was greater than 1500% of the control. This finding is significant for two reasons. First, as shown in fig. 2F, the increase in muscle mass after overload is not proportional to the average increase in fiber CSA. In fact, muscle mass appears to increase by-80% before an increase in mean fiber CSA is observed. However, we did not observe a significant increase in the number of fibers during this period. These data indicate that the majority of the increase in muscle mass that occurred after 14 days of functional overload was not due to an increase in mean fiber CSA. In the metatarsal muscle, there is a very large area of fibers and other areas of relatively small fibers. It is possible that the regional differences in the fibers CSA offset any significant effect on the average fiber area after overload. However, muscles may also grow in other ways. After the soleus and gastrocnemius muscles were removed, the ankle of the rat was held in a more dorsiflexed position, which is expected to increase the resting length of the metatarsals. We have preliminary data showing that one consequence of ankle joint positional deviation is an increase in the length of the plantar muscle of the foot of about 10%. Others have recently proposed similar recommendations in mice (10). These data indicate that functional overload may lead to increased mass of the plantar muscles of the foot due in part to the addition of muscle segments in tandem.

The finding that the best group had a mean fiber CSA increase of 61.5% compared to 4% in the control group was also significant for the magnitude of the hypertrophic difference obtained with the natural product mixture. Other treatments that increase muscle hypertrophy, such as depletion of leucine-rich protein, have an effect size of about 5% (3). These data indicate that the mechanism underlying the action of the natural product mixture is evident in skeletal muscle hypertrophy and may be rate limiting. One proposed limitation to skeletal muscle hypertrophy in mice and humans is the ability of protein synthesis; i.e., ribosome mass (8, 11, 18). To determine whether there is an increase in ribosome mass in animals fed the natural product mixture, we measured total RNA in muscle. Contrary to our hypothesis, total RNA tends to increase less for natural product mixtures compared to vehicle controls. To support this observation, the rRNA spacers (ITS 1 and 5 'ets) showed the same pattern, with a variation of 5' ets reaching statistical significance. These data indicate that even though hypertrophy is increased by the natural product mixture, the improvement is not a result of increased translational capacity.

One possible explanation for the apparent increase in muscle protein without concomitant increase in ribosome mass is an increase in translation efficiency. Although there have been many recent works on the acetylation of ribosomal proteins and the effect of translation efficiency, early works showed that, after hepatectomy, when the protein synthesis rate is increased to regenerate tissues, acetylation of ribosomal proteins precedes the protein synthesis reaction (12). This indicates that acetylation of ribosomal proteins can increase translation efficiency. Choudhury and colleagues identified 75 ribosomal proteins, which were acetylated at a minimum of 136 positions (4). For mitochondrial ribosomes, proteins MRPL10 and 19 were deacetylated by the mitochondrial SIRT3 SIRT (19). When SIRT3 is overexpressed, MRPLs 10 and 19 become deacetylated, which corresponds to a decrease in protein synthesis. When SIRT3, which is catalytically inactive, was used, there was no change in acetylation or protein synthesis. In addition, both acetylation and protein synthesis were increased when SIRT3 was targeted with shRNA (19). Finally, the isolation of ribosomes from the liver of SIRT3 knockout mice showed more protein synthesis per ribosomal protein unit (19). Taken together, these data suggest that sirtuins can deacetylate ribosomal proteins, which corresponds to a decrease in translation efficiency. Consistent with these data, we show that our proposed inhibition of SIRT1 of the natural product mixture tended to increase acetylation of both ribosomal proteins, which was associated with a greater change in (puromycin-like increase) protein synthesis relative to total RNA or ribosomal biogenesis (5' ets), suggesting increased translation efficiency.

In addition to acetylation of ribosomal proteins, the cell size regulator S6K1 can also be acetylated (6, 7), which reduces its ability to be phosphorylated by mTORC1 (9) and its ability to phosphorylate ribosomal proteins S6 in mesangial cells (5, 9). Deacetylation of S6K1 may be catalyzed by SIRT1 or SIRT2 (9). Thus, inhibition of SIRT1 is expected to increase S6K1 acetylation and decrease Thr389 phosphorylation. Interestingly, we observed a trend of simultaneous decrease in S6K1 acetylation and phosphorylation in muscles treated with the putative SIRT1 inhibitor for 14 days. When attempting to correct our data with the existing literature, it is important to note that in previous studies, the effect of SIRT1 on S6K1 acetylation occurred in cultures after 3 hours of treatment with the sirtuin inhibitor nicotinamide (9). It is also important to note that many in vitro studies are particularly concerned with acetylation of S6K1 at lysines 427, 484, 485 and 493. In contrast, current work observed complete acetylation of S6K1 after immunoprecipitation. Longer sirtuin inhibition in the presence of growth stress may lead to acetylation of 427, 484, 485, and 493, but deacetylation at other sites leads to the net deacetylation observed in this study.

Conclusion

Using in vitro assays, we have identified several natural products that are capable of inhibiting SIRT1 activity. By combining three of these inhibitors in vivo, we were able to develop a model of how different combinations contribute to muscle hypertrophy after overload. When this model was validated, we found that the optimal combination of natural products could significantly increase muscle hypertrophy in response to load, even though it significantly reduced ribosome biogenesis and tended to reduce the increase in ribosome mass that occurs with overload. This suggests that the natural products described herein may increase ribosomal efficiency through acetylation of ribosomal proteins, resulting in greater skeletal muscle hypertrophy.

Reference to the literature

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2.Celis-Morales CA,Welsh P,Lyall DM,Steell L,Petermann F,Anderson J,Iliodromiti S,Sillars A,Graham N,Mackay DF,Pell JP,Gill JMR,Sattar N,Gray SR.Associations of grip strength with cardiovascular,respiratory,and cancer outcomes and all cause mortality:prospective cohort study of half a million UK Biobank participants.doi:10.1136/bmj.k1651.

3.Cermak NM,Res PT,de Groot LC,Saris WH,van Loon LJ.Protein supplementation augments the adaptive response of skeletal muscle to resistance-type exercise training:a meta-analysis.Am J Clin Nutr 96:1454–64,2012.

4.Choudhary C,Kumar C,Gnad F,Nielsen ML,Rehman M,Walther TC,Olsen J V,Mann M.Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions[Online].science.sciencemag.org/[2 Jul.2019].

5.Das F,Maity S,Ghosh-Choudhury N,Kasinath BS,Ghosh Choudhury G.Deacetylation of S6 kinase promotes high glucose–induced glomerular mesangial cell hypertrophy and matrix protein accumulation.(2019).doi:10.1074/jbc.RA118.007023.

6.Fenton TR,Gwalter J,Cramer R,Gout IT.S6K1 is acetylated at lysine 516 in response to growth factor stimulation.Biochem Biophys Res Commun 398:400–405,2010.

7.Fenton TR,Gwalter J,Ericsson J,Gout IT.Histone acetyltransferases interact with and acetylate p70 ribosomal S6 kinases in vitro and in vivo.Int J Biochem Cell Biol 42:359–366,2010.

8.Figueiredo VC,Mccarthy JJ.Regulation of Ribosome Biogenesis in Skeletal Muscle Hypertrophy.(2019).doi:10.1152/physiol.00034.2018.

9.Hong S,Zhao B,Lombard DB,Fingar DC,Inoki K.Cross-talk between Sirtuin and Mammalian Target of Rapamycin Complex 1(mTORC1)Signaling in the Regulation of S6 Kinase 1(S6K1)Phosphorylation.(2014).doi:10.1074/jbc.M113.520734.

10.Jorgenson KW,Hornberger TA.The overlooked role of fiber length in mechanical load-induced growth of skeletal muscle.Exerc Sport Sci Rev 47:258–259,2019.

11.Kirby TJ,Lee JD,England JH,Chaillou T,Esser KA,Mccarthy JJ,Tj K,Lee CT,Ka E,Mccarthy JJ.Blunted hypertrophic response in aged skeletal muscle is associated with decreased ribosome biogenesis.J Appl Physiol 119:321–327,2015.

12.Liew CC,Gornall AG.Acetylation of Ribosomal Proteins in Regenerating Rat Liver[Online].pdfs.semanticscholar.org/0841/77fe07d19736f0e287aa1bdbd8ac23b9c5ff.pdf[2 Jul.2019].

13.Pandey KB,Rizvi SI.Plant polyphenols as dietary antioxidants in human health and disease.Oxid Med Cell Longev 2:270–8,[date unknown].

14.Rantanen T,Harris T,Leveille SG,Visser M,Foley D,Masaki K,Guralnik JM.Muscle Strength and Body Mass Index as Long-Term Predictors of Mortality in Initially Healthy Men[Online].academic.oup.com/biomedgerontology/article-abstract/55/3/M168/2947973[5 Jul.2019].

15.Ruiz JR,Sui X,Lobelo F,Morrow JR,Jackson AW,

M,Blair SN.Association between muscular strength and mortality in men:prospective cohort study.Br Med J 337:a439,2008.

16.Solomon JM,Pasupuleti R,Xu L,Mcdonagh T,Curtis R,Distefano PS,Huber LJ.Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage.Mol Cell Biol 26:28–38,2006.

17.Srikanthan P,Karlamangla AS.Muscle Mass Index As a Predictor of Longevity in Older Adults.Am J Med 127:547–553,2014.

18.Stec MJ,Kelly NA,Many GM,Windham ST,Tuggle SC,Bamman MM,Bamman MM.First published February 9.Am J Physiol Endocrinol Metab 310:652–661,2016.

19.Yang Y,Cimen H,Han M-J,Shi T,Deng J-H,Koc H,Palacios OM,Montier L,Bai Y,Tong Q,Koc EC.NAD+-dependent Deacetylase SIRT3 Regulates Mitochondrial Protein Synthesis by Deacetylation of the Ribosomal Protein MRPL10.(2009).doi:10.1074/jbc.M109.053421.

Table 1: SIRT1 inhibitor screening results for those compounds described in the examples. The table shows the name of each compound, the percent inhibition at 5 μ M or 50 μ M, and the molecular class to which each compound belongs.

Name of Compound	50μM	5μM	Types of
				Tripterine	100	101	Quinone methide
Tripterine dihydrogenum	99	84	Quinone
				Epigallocatechin-3-monogallate	99	72	Polyphenols
Epicatechin mono gallate	99	66	Flavonoid compounds

Table 2: validation of inhibitor Compounds and dosages used in the study

List of doses (mg/kg/day) of each inhibitor used in the validation study

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

Claims

1. A method of enhancing skeletal muscle growth in a mammal undergoing muscle loading comprising administering to the mammal a composition comprising a therapeutically effective amount of: (i) One or more catechins and (ii) tripterine or a derivative thereof.

2. The method of claim 1, wherein the one or more catechins comprises epicatechin mono-gallate or epigallocatechin-3-mono-gallate.

3. The method of claim 2, wherein the one or more catechins includes epicatechin mono-gallate and epigallocatechin-3-mono-gallate.

4. The method of any one of claims 1-3, wherein the tripterine derivative is dihydrotripterine.

5. The method of any one of claims 1-4, wherein the composition comprises epicatechin mono-gallate, epigallocatechin-3-mono-gallate, and celastrol.

6. The method of any one of claims 1 to 5, wherein the composition results in an increase in muscle fiber cross-sectional area of at least one skeletal muscle in a mammal.

7. The method of any one of claims 1 to 6, wherein the composition does not substantially alter the body weight or heart or liver weight of the mammal.

8. The method of any one of claims 1 to 7, wherein the composition reduces SIRT1 activity in one or more muscles of a mammal.

9. The method of any one of claims 1 to 8, wherein the composition increases acetylation of one or more ribosomal proteins in one or more muscles of the mammal.

10. The method of any one of claims 1-9, wherein the composition is administered orally to the mammal.

11. The method of claim 10, wherein the composition is formulated as a nutritional supplement or food additive.

12. The method of claim 11, wherein the nutritional supplement or food additive is a pill, tablet, capsule, liquid, powder, energy bar, protein bar, or gum.

13. The method of any one of claims 1 to 12, wherein the mammal is a human.

14. The method of any one of claims 1 to 13, wherein the composition is formulated and administered such that the mammal receives about 0.7-1.3 mg/kg/day of epicatechin mono gallate.

15. The method of claim 14, wherein the composition is formulated and administered such that the mammal receives about 0.7 mg/kg/day of epicatechin mono-gallate.

16. The method of any one of claims 1 to 15, wherein the composition is formulated and administered such that the mammal receives about 6-20 mg/kg/day of epigallocatechin-3-mono-gallate.

17. The method of claim 16, wherein the composition is formulated and administered such that the mammal receives about 20 mg/kg/day of epigallocatechin-3-mono-gallate.

18. The method of any one of claims 1-17, wherein the composition is formulated and administered such that the mammal receives about 0.2-0.5 mg/kg/day of celastrol.

19. The method of claim 18, wherein the composition is formulated and administered such that the mammal receives about 0.5 mg/kg/day of celastrol.

20. The method of any one of claims 1-19, wherein the composition is formulated and administered such that the mammal receives about 0.7 mg/kg/day of epicatechin mono-gallate, about 20 mg/kg/day of epigallocatechin-3-mono-gallate, and about 0.5 mg/kg/day of celastrol.

21. The method of any one of claims 1-20, wherein the composition is formulated and administered such that the relative weight ratios of each of the epicatechin mono-gallate, epigallocatechin-3-mono-gallate and celastrol received by the mammal are from about 0.7.

22. The method of any one of claims 1-21, wherein the composition is formulated and administered such that the relative molar ratio of each of the epicatechin mono-gallate, epigallocatechin-3-mono-gallate and tripterine received by the mammal is from about 1.6.

23. The method of any one of claims 1 to 22, further comprising increasing caloric intake and/or muscle growth promoting amino acid intake of the mammal concurrently with muscle loading and administration of the composition.

24. The method of any one of claims 1 to 23, wherein the composition further comprises leucine, branched chain amino acids, or proteins with high leucine content.

25. A composition for enhancing muscle growth in a mammal undergoing muscle loading, the composition comprising a therapeutically effective amount of (i) one or more epicatechin and (ii) tripterine or a derivative thereof.

26. The composition of claim 25, wherein the one or more epicatechin includes epicatechin mono-gallate or epigallocatechin-3-mono-gallate.

27. The composition of claim 26, wherein the one or more epicatechin includes epicatechin mono-gallate and epigallocatechin-3-mono-gallate.

28. The composition of any one of claims 25-27, wherein the tripterine derivative is dihydrotripterine.

29. The composition of any one of claims 25-28, wherein the composition comprises epicatechin mono-gallate, epigallocatechin-3-mono-gallate, and celastrol.

30. The composition of any one of claims 25-29, wherein the composition is formulated for oral administration.

31. The composition of claim 30, wherein the composition is formulated as a nutritional supplement or a food additive.

32. The composition of claim 31, wherein the nutritional supplement or food additive is a pill, tablet, capsule, liquid, powder, energy bar, protein bar, or gum.

33. The composition of claim 32, further comprising leucine, branched chain amino acids, or proteins with high leucine content.

34. The composition of any one of claims 25 to 33, wherein the composition is formulated such that the mammal receives about 0.7-1.3 mg/kg/day of epicatechin mono-gallate.

35. The composition of claim 34, wherein the composition is formulated such that the mammal receives about 0.7 mg/kg/day of epicatechin mono-gallate.

36. The composition of any one of claims 25-35, wherein the composition is formulated such that the mammal receives about 6-20 mg/kg/day of epigallocatechin-3-mono-gallate.

37. The composition of claim 36, wherein the composition is formulated such that the mammal receives about 20 mg/kg/day of epigallocatechin-3-mono-gallate.

38. The composition of any one of claims 25-37, wherein the composition is formulated such that the mammal receives about 0.2-0.5 mg/kg/day of celastrol.

39. The composition of claim 38, wherein the composition is formulated such that the mammal receives about 0.5 mg/kg/day of celastrol.

40. The composition of any one of claims 25-39, wherein the composition is formulated such that the mammal receives about 0.7 mg/kg/day of epicatechin mono-gallate, about 20 mg/kg/day of epigallocatechin-3-mono-gallate, and about 0.5 mg/kg/day of tripterine.

41. The composition of any one of claims 25-40, wherein the relative weight ratio of epicatechin mono-gallate, epigallocatechin-3-mono-gallate, and celastrol, respectively, in the composition is about 0.7.

42. The composition of any one of claims 25-41, wherein the relative molar ratio of each of epicatechin mono-gallate, epigallocatechin-3-mono-gallate and tripterine in the composition is about 1.6.